The focus of this proposal is the nicotinic-receptor superfamily of ion channels. Our long-term goal is to elucidate how the biophysical properties of these receptor-channels shape their function under both normal and pathological conditions. To this end, we will apply a multi-faceted approach that includes single-channel and ensemble electrophysiology, protein engineering, X-ray crystallography and theoretical calculations. Most of the experiments will be conducted on the muscle nicotinic acetylcholine receptor (AChR) and its bacterial counterparts, ELIC and GLIC. Because of its large single-channel conductance, robust stoichiometry, tractable kinetics and high expression levels in heterologous expression systems, the muscle AChR remains an unparalleled model for the entire superfamily. Furthermore, throughout the years, research on this particular receptor has provided answers to fundamental questions on the physics, chemistry and biology of ion channels that cannot be addressed with (probably) any other channel. In this application, we propose to address the three pillars of ion-channel function, namely, ion conduction, gating, and modulation of gating. Our three Specific Aims are: 1) To characterize the ring of glutamates in the charge selectivity filter of the AChR in terms of number of total negative charges and acid-base behavior; 2) To identify the nature of the gate(s) and its rearrangement upon channel opening, closing and desensitization; and 3) To characterize the regions of the AChR hypothesized to be involved in the metabolic and intramembrane modulation of function. Because of its role as a paradigmatic neurotransmitter-gated ion channel, the knowledge derived from the proposed experiments on the muscle AChR is poised to have a broad impact on our understanding of postsynaptic receptors and fast synaptic transmission in general. It should also be emphasized that AChR-mediated neurotransmission supports not only motor, but also, autonomic and cognitive function. As a result, in addition to being compromised in myasthenic syndromes, cholinergic neurotransmission is also impaired in cardiovascular diseases and neurodegenerative disorders such as Alzheimer's disease and schizophrenia. Even more broadly, AChRs are also expressed in the airways, keratinocytes, lymphocytes and endothelial cells where they are mediators of cell differentiation, proliferation and resistance to apoptosis, and hence, of cancer.